Bacteria exhibit extreme morphological diversity, a fact known since they were first visualized over 300 years ago. Yet only in the last 5-7 years has it become clear that these differences are much more than cosmetic. Instead, cell shape is of fundamental and medical importance, contributing to bacterial survival and virulence by influencing nutrient uptake, cell-to-surface attachment, motility, differentiation, and resistance to predation and host immune assaults. Nor is morphology static: bacteria invest significant resources to manipulate their shapes to cope with changes in growth rate and nutritional status and to respond to antibiotics and other environmental stresses. In short, morphology is not a frivolous or neutral characteristic, but plays crucial roles in bacterial cell biology, ecology and pathogenesis. Despite a recent renaissance in such studies, we understand surprisingly little about how cells create and maintain their shapes. What we do know is that most bacteria are protected by a peptidoglycan cell wall and that the way this wall is synthesized determines cell shape. Our long-term goal is to understand the structure, synthesis, regulation and functional implications of peptidoglycan and the enzymes that create and modify it. To that end, we propose to extend our understanding of bacterial physiology and morphology by pursuing the following Aims. Aim 1] Identify new morphological mechanisms and regulators by applying newly adapted methods for enriching shape mutants via fluorescence- activated cell sorting (FACS). One reason so little is known about these matters is because almost all morphological regulatory agents were discovered accidently. Here, we will use FACS as a genetic tool to enrich, isolate and study morphological mutants in a directed search for new mechanisms that regulate bacterial shape. Aim 2] Identify and characterize the mechanisms required for de novo shape generation by employing a newly devised Spheroplast Recovery assay. Virtually everything known about cell wall synthesis and morphology involves mechanisms that preserve or extend pre-existing walls. Little or nothing is known about how cells re-create their shapes when their walls are damaged severely or removed altogether, as occurs when bacteria encounter host immune systems. We find that survival in these latter circumstances demands new mechanisms to support or supplant the classic maintenance pathways. Aim 3] Characterize critical FtsZ- peptidoglycan reactions, particularly those that will tell us: a) how periplasmic peptidoglycan and penicillin binding proteins control the geometry of the cytoplasmic Z ring during cell division; and b) how cytoplasmic FtsZ triggers the synthesis of peptidoglycan during the critical transition from cell elongation to division. These poorly-understood interactions determine the integrity, shape and propagation of bacterial cells. In summary, these new tools and approaches will enable us to investigate, faster and in greater depth, the numerous mysteries that still obscure our understanding of some of the most basic issues in bacterial physiology. The work will also serve as a model for similar morphological investigations in other organisms.